2.
medical specialties and that 2) vigilance as well
as cognitive performance is more compromised
after 24 hours overnight on-call duty compared
to night shift.
METHODS This pilot study included 38 neurology residents
(19 women and 19 men, mean age 30 Ϯ 2 years) recruited from the
Department of Neurology at the University Hospital Carl Gustav
Carus, Dresden, Germany. We screened residents for exclusion cri-
teria such as current use of medication known to affect the sleep/
wake cycle or daytime alertness, current psychiatric illness, and sleep
disorder diagnosis. We then stratified eligible residents according to
their working schedule into 3 groups: group 1, 24 hours overnight
on-call duty; group 2, night shift; and group 0, regular day shift
(control).
Definitions of groups. Residents in group 1 (24 hours over-
night on-call duty) performed their regular shift from 8 AM–4
PM followed by overnight call until 8 AM the next day. During
their overnight duty, they were allowed to sleep but there was no
scheduled coverage. Residents have usually 3 to 4 overnight calls
per month. Residents in group 2 (night shift) worked at the
intensive care unit for 7 consecutive days daily from 8 PM to 8 AM
the following morning. Afterwards they had 1 week off. Sleeping
was not permitted during their shifts. From experience, residents
have on average 2 admissions and 30 internal and 3 outpatient
consultations per night. Night shift rotation was every 5 to 6
weeks over a period of 1 year. Residents in group 0 (day shift)
regularly worked from 7:30 AM to 3:30 PM. However, residents
on day shift frequently work overtime.
Standard protocol approvals, registrations, and patient con-
sents. The Ethics Committee on human experimentation of the
Dresden University of Technology approved the study and the in-
vestigation conformed with the principles outlined in the Declara-
tion of Helsinki. We informed all participating residents about the
objectives and procedures of the study and obtained written in-
formed consent prior to their inclusion. The serial data collection
was performed at the Autonomic and Neuroendocrinological Labo-
ratory of the University Clinic. We measured all residents before 9
AM directly after their (night) shift rotation or just before day shift
commenced (controls). The assistant performing the measurements
was blinded. We measured objective sleepiness by Pupillography
Sleepiness Test (PST) and cognitive performance by Paced Audi-
tory Serial Addition Test (PASAT). We instructed the residents to
abstain from drinking alcoholic beverages, smoking, and drinking
coffee for at least 4 hours before the measurements. The residents
rated their sleepiness on a 5-point Likert scale based on the state-
ment “Currently I feel.” We also recorded the number of hours slept
in the previous 24 hours and assessed the perceived recovery effect
due to sleep on a 5-point Likert scale. All measurements were serially
repeated up to a maximum of 13 times. Measurement frequency,
however, varied between individuals according to the rotation
schedules (4.8 Ϯ 3.3).
Pupillary Sleepiness Test. We performed the PST (AMTech,
Dossenheim, Germany) in a quiet and darkened room after an ini-
tial dark-adapting phase of 15 minutes. During PST, residents wore
goggles equipped with infrared light transmitting filter glasses im-
pervious to visible light. They were seated on a comfortable chair
and head position was adjusted by a chin rest fixed on a table. An
infrared video camera was fixed at a distance of 70 cm from the
examination subject. We instructed the clinicians to maintain fixa-
tion on a set of infrared light-emitting diodes. We then recorded
spontaneous pupillary oscillations over a period of 11 minutes by
infrared video pupillography and evaluated the recording by 25-Hz
real-time analysis as published elsewhere.5
Pupillary unrest index
(PUI) is a measure of pupillomotor hippus in darkness and calcu-
lated as an integrated sum of slow movements of the pupillary mar-
gin during the measurement period.6
This value is usually low in
alertness and increases with progressive sleepiness. We also calcu-
lated the mean pupil diameter over the entire recording period of 11
minutes. During sleepiness the initial diameter is reduced and the
mean pupil size falls below the initial diameter toward the end of the
measurement.
Paced Auditory Serial Addition Test. The validated and
computer-aided PASAT allows for measuring the capacity and
velocity of information processing within the auditory-verbal do-
main (cognitive performance).7,8
The test system entails the sub-
ject to continuously add the last 2 numbers of consecutive series
and to announce the sum aloud. Numbers from 1 to 9 are an-
nounced acoustically in random order by a PC with the screen
remaining dark. To avoid practice effects, clinicians were trained
on the PASAT at least 3 times before commencing the study. We
applied the 60-item short version of the test (maximal score of
60). Lower scores (small numbers of correct answers) indicate
worse cognitive performance.
Statistical analysis. We used the SPSS software package ver-
sion 16.0 for Windows (SPSS Inc., Chicago, IL) for all statistical
evaluation. Data are presented as median and 25th–75th percen-
tile unless otherwise stated. Owing to the small sample size we
assumed non-Gaussian distribution, and hence applied nonpara-
metric tests with Bonferroni correction for comparing groups.
Spearman correlation coefficients were calculated. A two-tailed
p Ͻ 0.05 was regarded as the level of significance.
RESULTS Before we started the comparison among
the 3 groups, we assessed the strength of association
between the first measurement and the mean of serial
Table Sleepiness and cognitive performance of neurology residents by type of night duty
Parameters Overnight call (n ‫؍‬ 17) Night shift (n ‫؍‬ 6) Control (n ‫؍‬ 15) ␹2
(p*)
Pupil diameter (mm) 7.51 (6.25–7.75) 7.23 (5.18–7.54) 7.21 (6.61–8.00) 0.59 (0.744)
Pupillary unrest index (mm/min) 7.00 (4.96–9.44)a
10.34 (7.78–15.07)a
4.72 (3.86–5.09)b
12.88 (0.002)
PASAT score† 56 (49–58) 51 (42–58) 54 (48–56) 0.67 (0.715)
Self-stated sleepiness‡ 2.3 (1.7–2.7)a
2.6 (2.2–3.1)a
1.0 (0–1.0)b
20.24 (Ͻ0.001)
Data are median (interquartile range). Unequal superscript letters indicate significant differences (Mann-Whitney U test).
*Kruskal-Wallis test.
†Paced Auditory Serial Addition Test (number of correct answers/60).
‡Categorical value.
e100 Neurology 73 November 24, 2009

3.
measurements. We revealed significant correlations
with r values ranging from 0.763 to 0.904 (p Ͻ
0.001). Based on these results, we continued our
analysis using the mean values.
PUI and self-stated sleepiness (SSS) were signifi-
cantly affected by type of night duty while pupil diame-
ter and the PASAT score remained unaffected (table). It
appeared that PUI and SSS were significantly higher
after the night shift and the 24 hours overnight on-call
duty compared to a normal night at home. Residents
after night shift and 24 hours overnight on-call duty did
not differ with respect to sleepiness measures.
Neurology residents on 24 hours overnight on-
call duty had slept on average 4.3 (2.8–4.6) hours
(midshift nap) in the last 24 hours, which was signif-
icantly less compared to their colleagues on night
shift (5.9 [4.9–7.0] hours, p ϭ 0.006) or on day shift
(controls) (6.5 [6.0–7.0] hours, p Ͻ 0.001). The
longest sleeping phase during 24 hours overnight on-
call duty was 3.0 (2.0–3.8) hours. Residents had on
average a mean (minimum–maximum) of 2 (1–3)
admissions, 2 (0–7) consultations, and 3 (1–5) tele-
phone inquiries during overnight on-call duty.
Figure 1 illustrates the proportion of respective
responses to the statement “Currently I feel . . .” Fig-
ure 2 depicts the self-stated recovery effect due to
sleep in the 24 hours preceding the examination.
Correlation analyses did not reveal any association
between the PUI and the PASAT score. However, the
PUI increased (r ϭ 0.507, p ϭ 0.001) and the PASAT
score decreased (r ϭ Ϫ0.335, p ϭ 0.04) with increased
SSS in the total sample. The perceived level of sleepiness
decreased as the number of sleeping hours in the past 24
hours increased (r ϭ Ϫ0.527, p ϭ 0.001). The above
associations could not be confirmed in subgroups (p Ͼ
0.05).
DISCUSSION Rotating shift work in clinics to pro-
vide 24-hour patient care has come increasingly under
scrutiny due to negative effects associated with sleep
loss, fatigue, and circadian disruption.9,10
Although
night shift and 24 hours overnight on-call duty consid-
erably differ in terms of number of working hours, per-
mission for midshift naps, and rotation frequency, their
effect on sleepiness and cognitive performance has never
been distinguished. We hypothesized that residents on
24 hours call rotation would be more affected by sleep
loss due to a longer and more irregular working sched-
ule. We investigated this hypothesis in neurology resi-
dents of a large university clinic. This specialty group is
often underrated in terms of heaviness and intensity of
labor and therefore has never been investigated in sleep-
iness studies. Importantly, previous sleepiness studies in
selected medical specialties explicitly emphasized that
results must not be extrapolated to other medical spe-
cialty groups.3,4,11
Although we could not verify the
above hypothesis, our results clearly demonstrate that
sleepiness is a common problem among neurology resi-
dents undergoing night shift and 24 hours overnight
on-call duty.
We additionally demonstrated that vigilance mea-
sured by PST is in good agreement with SSS. This
finding corresponds with previous studies suggesting
the PST as a valid and objective tool to detect sleepi-
ness in healthy subjects.6
The lack of significant performance decrements in
sleep-deprived neurology residents vs controls is intrigu-
ing since an association between performance and acute
sleep deprivation was found in previous studies.2,4,12
Differences in study design, medical specialty, and
methods for vigilance testing limit comparisons across
studies. However, one study also failed to show a signif-
icant performance decrement on the complex PASAT
test in sleep-deprived normal subjects.13
The investiga-
tors concluded that university-based research may func-
Figure 1 Self-stated sleepiness of neurology residents by type of night duty
Figure 2 Self-stated recovery effect due to sleep in the past 24 hours
Neurology 73 November 24, 2009 e101

16.
Articles
www.thelancet.com Vol 373 March 28, 2009 1105
Long-term risk of epilepsy after traumatic brain injury in
children and young adults: a population-based
cohort study
Jakob Christensen, Marianne G Pedersen, Carsten B Pedersen, Per Sidenius, Jørn Olsen, MogensVestergaard
Summary
Background The risk of epilepsy shortly after traumatic brain injury is high, but how long this high risk lasts is
unknown. We aimed to assess the risk of epilepsy up to 10 years or longer after traumatic brain injury, taking into
account sex, age, severity, and family history.
Methods We identiﬁed 1605216 people born in Denmark (1977–2002) from the Civil Registration System. We obtained
information on traumatic brain injury and epilepsy from the National Hospital Register and estimated relative risks
(RR) with Poisson analyses.
Findings Risk of epilepsy was increased after a mild brain injury (RR 2·22, 95% CI 2·07–2·38), severe brain injury
(7·40, 6·16–8·89), and skull fracture (2·17, 1·73–2·71). The risk was increased more than 10 years after mild brain
injury (1·51, 1·24–1·85), severe brain injury (4·29, 2·04–9·00), and skull fracture (2·06, 1·37–3·11). RR increased
with age at mild and severe injury and was especially high among people older than 15 years of age with mild (3·51,
2·90–4·26) and severe (12·24, 8·52–17·57) injury. The risk was slightly higher in women (2·49, 2·25–2·76) than in
men (2·01, 1·83–2·22). Patients with a family history of epilepsy had a notably high risk of epilepsy after mild (5·75,
4·56–7·27) and severe brain injury (10·09, 4·20–24·26).
Interpretation The longlasting high risk of epilepsy after brain injury might provide a window for prevention of
post-traumatic epilepsy.
Funding Danish Research Agency, P A Messerschmidt and Wife’s Foundation, Mrs Grethe Bønnelycke’s Foundation.
Introduction
Traumatic brain injury raises the risk of epilepsy,1
but
little is known about the duration of the increased risk
and the factors that modify the risk, especially in children
and young adults.2
In hospital-based case series, the risk
of epilepsy 1–2 years after moderate to severe brain injury
is related to some CT or MRI ﬁndings and is high in
people who had had neurosurgical procedures.2–6
In a
population-based study, age in people who had traumatic
brain injury at age 65 years or older and time since and
severity of injury were signiﬁcant risk factors for epilepsy,1
but only a few studies have included children and young
adults.1–4
In some of these studies,2,4
acute seizures in the
ﬁrst week after brain injury were associated with a high
risk of epilepsy. Studies of epilepsy related to level of
consciousness (eg, assessed with the Glasgow coma
scale) and duration of post-traumatic amnesia after brain
injury have given conﬂicting results.1,3,4
No eﬀective prophylaxis for epilepsy after traumatic
brain injury is available, and trials with preventive drug
have been discouraging.7
However, better information
about prognostic factors might help the development of
new prevention strategies and treatment.5
We studied the risk of epilepsy in a large
population-based cohort of children and young adults
and considered time since injury, sex, age, severity, and
family history of epilepsy.
Methods
Study population
We used data from the Danish Civil Registration System
(CRS)8
to identify all people born in Denmark between
Jan 1, 1977, and Dec 31, 2002. All liveborn children and
new residents in Denmark are assigned a unique
personal identiﬁcation number (CRS number) together
with information on vital status, emigration from
Denmark, and CRS numbers of mothers, fathers, and
siblings. The CRS number links individual information
in all national registries and provides identiﬁcation of
family members and links parents with their children.
Identity of individuals in the study was blinded to the
investigators, and the study did not involve contact with
individual patients. The study therefore did not need
approval from the ethics committee according to Danish
laws, but the project was approved by the Danish Data
Protection Agency.
Data collection
Information about brain injury and epilepsy was obtained
from the Danish National Hospital Register,9
which
contains information on all discharges from Danish
hospitals since 1977; outpatients have been included in the
register since 1995. All treatment is free of charge for
Danish residents. Patients admitted to the only private
epilepsy hospital in Denmark are also recorded in the
Lancet 2009; 373: 1105–10
Published Online
February 23, 2009
DOI:10.1016/S0140-
6736(09)60214-2
See Comment page 1060
Department of Neurology,
Aarhus University Hospital,
Aarhus, Denmark
(J Christensen MD,
P Sidenius MD); Department of
Clinical Pharmacology
(J Christensen) and National
Centre for Register-based
Research (M G Pedersen MSc,
C B Pedersen MSc), University of
Aarhus, Denmark; Southern
California Injury Prevention
Research Centre (SCIPRC),
School of Public Health, UCLA,
CA, USA (J Olsen MD),
Department of Epidemiology
(J Olsen) and Department of
General Practice
(MVestergaard MD), Institute
of Public Health, University of
Aarhus, Aarhus, Denmark
Correspondence to:
Dr Jakob Christensen,
Department of Neurology,
Aarhus University Hospital,
Norrebrogade 44,
DK-8000 Aarhus C, Denmark
jakob@farm.au.dk

17.
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1106 www.thelancet.com Vol 373 March 28, 2009
Danish National Hospital Register. Specialists in neurology
working in private outpatient clinics also treat patients
with epilepsy, but these contacts are not recorded in the
Danish National Hospital Register.
Diagnostic information in the National Hospital
Register is based on the International Classiﬁcation of
Diseases, 8th revision (ICD-8) from 1977–93, and ICD-10
from 1994–2002.
Cohort members, their parents and siblings were
classiﬁed with epilepsy if they had been hospitalised or in
outpatient care with a diagnosis of epilepsy (ICD-8: 345;
ICD-10: G40,G41).10–13
By use of the CRS numbers, we
linked parents and siblings registered with an epilepsy
diagnosis in the National Hospital Register. A person was
recorded as having a family history of epilepsy if the date
of ﬁrst epilepsy diagnosis in a parent or sibling preceded
their epilepsy diagnosis.
Cohort members were classiﬁed with mild brain injury
(concussion: ICD-8 850.99; ICD-10 S06.0), severe brain
injury (structural brain injury: ICD-8 851.29-854.99;
ICD-10S06.1-S06.9),orskullfracture(ICD-8800.99–801.09,
803.99; ICD-10: S02–S02.1, S02.7, S02.9), respectively, if
they had been admitted or been in outpatient care with the
relevant diagnosis.14,15
Time of onset of epilepsy and brain
injury was deﬁned as the ﬁrst day of the ﬁrst contact to the
hospital with the relevant diagnosis.
The deﬁnition of mild brain injury (concussion) in
Denmark is based on the deﬁnition given by the American
Congress of Rehabilitation Medicine.16
The diagnostic
criteria include a relevant direct trauma against the head
manifesting with changed brain function (ie, loss of
consciousness, amnesia, confusion/disorientation, or
focal [temporary] neurological deﬁcit). Severity of mild
brain injury should not include loss of consciousness
longer than 30 min, a Glasgow coma scale of 13 or less
after 30 min, or post-traumatic amnesia longer than 24 h.17
Severe brain injury (structural brain injury) includes
brain contusion or intracranial haemorrhage. Skull
fracture liable to be associated with disruption of brain
function can occur alone or be associated with other types
of brain injury and usually requires veriﬁcation with a
radiograph or CT. Brain injuries recorded in the same
patient within 14 days were categorised as the same event
according to the hierarchy of brain injury—severe brain
injury, skull fracture, and mild brain injury. For each type
of brain injury, we calculated the age at ﬁrst brain injury
(0–5, 5–10, 10–15, and ≥15 years), the length of ﬁrst
admission (0, 1–6, 7–13, 14–27, and ≥28 days), and time
since ﬁrst brain injury (0–6 months, 6 months to 1 year,
1–2, 2–3, 3–5, 5–10, and ≥10 years).
Statistical analyses
People were followed from birth until onset of epilepsy,
death, emigration from Denmark, or Dec 31, 2002,
whichever came ﬁrst. The incidence rate ratio (for these
analyses a good approximation of the relative risk, the
term used in this Article) of epilepsy was estimated by
Patients diagnosed
with epilepsy
New cases (per
1000 person-years)
Adjusted relative risk
(95% CI)
p value
Time (years) since mild brain injury
0·0–0·5 162 4·67 5·46 (4·67–6·37) <0·0001
0·5–1·0 78 2·37 2·91 (2·33–3·64) <0·0001
1·0–2·0 109 1·78 2·26 (1·87–2·73) <0·0001
2·0–3·0 99 1·79 2·33 (1·91–2·84) <0·0001
3·0–5·0 138 1·50 1·99 (1·68–2·36) <0·0001
5·0–10·0 154 1·14 1·56 (1·33–1·83) <0·0001
≥10·0 97 1·00 1·51 (1·24–1·85) <0·0001
No mild injury 16633 0·87 1·00 ··
Time (years) since severe brain injury
0·0–0·5 35 19·62 21·26 (15·25 to 29·62) <0·0001
0·5–1·0 19 11·52 13·45 (8·57 to 21·09) <0·0001
1·0–2·0 18 6·06 7·42 (4·68 to 11·79) <0·0001
2·0–3·0 11 4·26 5·40 (2·99 to 9·76) <0·0001
3·0–5·0 11 2·69 3·52 (1·95 to 6·35) <0·0001
5·0–10·0 15 3·22 4·40 (2·65 to 7·30) <0·0001
≥10·0 7 2·94 4·29 (2·04 to 9·00) 0·0001
No severe injury 17354 0·89 1·00 ··
Time (years) since skull fracture
0·0–0·5 6 2·90 2·96 (1·33 to 6·60) 0·0078
0·5–1·0 6 2·99 3·51 (1·58 to 7·83) 0·0021
1·0–2·0 13 3·38 4·30 (2·50 to 7·41) <0·0001
2·0–3·0 5 1·39 1·81 (0·75 to 4·35) 0·1845
3·0–5·0 9 1·36 1·78 (0·93 to 3·42) 0·0838
5·0–10·0 16 1·21 1·55 (0·95 to 2·54) 0·0781
≥10·0 23 1·46 2·06 (1·37 to 3·11) 0·0005
No fracture 17392 0·89 1·00 ··
Each form of injury led to a signiﬁcant (p<0·0001) increase in risk of epilepsy relative to people without brain injury.
Relative risk (RR) was adjusted for age and interaction with sex and calendar year. RR of epilepsy in people with brain
injury was modiﬁed by time since ﬁrst admission with brain injury for mild (p<0·0001) and severe (p<0·0001) brain
injury but not skull fracture (p=0·16).
Table 1: Time since ﬁrst admission with brain injury and relative risk (RR) of epilepsy
35
30
25
20
15
10
5
0
Relativeriskofepilepsy
0 1 2 3 4 5 6 7 8 9 ≥10
Years after injury
Mild brain injury
Severe brain injury
Skull fracture
Reference
Figure: Relative risk of epilepsy after brain injury in Denmark (1977–2002)

18.
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www.thelancet.com Vol 373 March 28, 2009 1107
log-linear Poisson regression18
with the GENMOD
procedure in SAS (version 8.1). Because incidence of
epilepsy depends on age, sex, and calendar year,10
all the
relative risks were adjusted for these factors. Age,
calendar year, age at ﬁrst brain injury, duration of ﬁrst
admission with brain injury, time since ﬁrst brain injury,
and history of epilepsy in a parent or sibling were time
dependent variables;19
all other variables were treated as
time independent. Age was categorised in quarter year
age levels from birth to the ﬁrst birthday, in 1 year age
levels from the ﬁrst birthday to the 20th birthday, and as
20–21 years and ≥22 years. Calendar year was categorised
in 1 year periods from 1977 to 2002. Likelihood ratio tests
were used to calculate p values and 95% CIs were
calculated by use of Wald’s test.19
The adjusted-score test20
suggested that the regression models were not subject to
overdispersion.
Role of the funding source
The sponsors had no role in the study design, data
collection, data analysis, data interpretation, or writing of
the Article. All authors had full access to the data and
approved the decision to submit the Article for publication
in The Lancet.
Results
We followed-up 1605216 for a total of
19527337 person-years. During this study period,
78572 people had at least one traumatic brain injury, and
in the same period, 17470 people developed epilepsy, of
whom 1017 had a preceding brain injury. Follow-up was
stopped before the end of the study period for
45677 people (2·9%) because of emigration from
Denmark (30362 [1·9%]) or death (15 315 [1·0%]).
Relative to no brain injury, the risk of epilepsy was two
times higher after mild brain injury (RR 2·22, 95% CI
2·07–2·38); seven times higher after severe brain injury
(7·40, 6·16–8·89); and two-times higher after skull
fracture (2·17, 1·73–2·71).
Tables 1–3 show the risk of epilepsy after brain injury
according to time since ﬁrst admission with brain
injury, age at ﬁrst brain injury, and duration of ﬁrst
hospital stay with brain injury.
The risk of epilepsy after mild (p<0·0001) and severe
(p<0·0001) brain injury was highest during the ﬁrst
years after injury, but remained high for more than
10 years after the injury as compared with people without
such a history (table 1, ﬁgure). For patients with skull
fractures, risk of epilepsy did not vary signiﬁcantly with
time since injury (p=0·16; table 1).
Brain injury was associated with an increased risk of
epilepsy in all age groups (table 2). The risk increased
with age for mild (p<0·0001) and severe (p=0·02) brain
injury and was highest among people older than
15 years at injury.
Patients who had a long duration of hospital stay with
severe brain injury (p<0·0001) and skull fracture
(p=0·02) had a notably high risk of epilepsy (table 3). For
people with mild brain injury there was no association
between duration of hospital stay and risk of epilepsy
(p=0·73; table 3).
Table 4 shows the relative risk of epilepsy after brain
injuries subdivided by family history of epilepsy. The
relative risk of epilepsy with a family history of the
disorder and mild brain injury is between what would
have been predicted from a multiplicative model
(3·37×2·24=7·54) and from an additive model
(3·37+2·24–1=4·61; table 4). The relative risk estimate
associated with severe brain injury and family history of
epilepsy of is almost the same as would have been
predicted from an additive model (3·35+7·81–1=10·16;
table 4). We had very few people with epilepsy with skull
fracture and a family history of epilepsy (table 4).
The relative risk of epilepsy after mild brain injury was
higher among women (2·49, 2·25–2·76) than among
men (2·01, 1·83–2·22; p=0·003). There was no
interaction with sex for patients with skull fractures
(p=0·59) or severe brain injury (0·22). We calculated the
risk of epilepsy for patients registered with brain injury
according to ICD-8 and ICD-10 (ie, patients diagnosed in
the time period 1977 to 1993 and 1994 to 2002,
respectively). For patients with mild brain injury, the risk
of epilepsy was lower in the ICD-8 period (RR 1·89,
1·71–2·10) than in the ICD-10 period (2·61, 2·37–2·87;
p<0·0001). For severe brain injury, the risk of epilepsy
was almost the same in the ICD-8 period (7·17,
Number of patients
with epilepsy*
New cases (per
1000 person-years)
Adjusted relative risk
(95% CI)
p value
Age (years) at mild brain injury
0–5 365 1·64 2·06 (1·86–2·29) <0·0001
5–10 243 1·56 2·12 (1·87–2·41) <0·0001
10–15 117 1·54 2·25 (1·88–2·71) <0·0001
≥15 112 2·03 3·51 (2·90–4·26) <0·0001
No mild injury 16633 0·87 1·00 ··
Age (years) at severe brain injury
0–5 51 6·26 7·20 (5·47–9·48) <0·0001
5–10 24 4·96 6·18 (4·14–9·23) <0·0001
10–15 11 3·56 4·91 (2·72–8·87) <0·0001
≥15 years 30 7·47 12·24 (8·52–17·57) <0·0001
No severe injury 17354 0·89 1·00 ··
Age (years) at skull fracture
0–5 52 1·53 1·95 (1·49–2·56) <0·0001
5–10 17 2·12 2·86 (1·78–4·60) <0·0001
10–15 5 1·81 2·55 (1·06–6·12) 0·0368
≥15 years 4 1·71 2·75(1·03–7·34) 0·0433
No skull fracture 17392 0·89 1·00 ··
Each form of injury led to a signiﬁcant (p<0·0001) increase in risk of epilepsy relative to people without brain injury.
Relative risk (RR) was adjusted for age and interaction with sex and calendar year. RR of epilepsy in people with brain
injury was modiﬁed by age at ﬁrst admission with brain injury for mild (p<0·0001) and severe (p=0·02) brain injury but
not skull fracture (p=0·55).
Table 2: Age at ﬁrst admission with brain injury and relative risk of epilepsy

19.
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1108 www.thelancet.com Vol 373 March 28, 2009
5·19–9·91) and the ICD-10 period (7·51, 6·02–9·38;
p=0·82). For skull fracture, the risks of epilepsy were
comparable in the ICD-8 period (2·00, 1·54–2·58) and
ICD-10 period (2·87, 1·85–4·46; p=0·17).
Discussion
As previously shown in studies smaller than ours,1,2,6,21
risk of epilepsy increased after brain injury in relation to
severity of brain injury. Risk was high for more than
10 years after the brain injuries even for mild brain
injury (concussion), a ﬁnding in contrast to that of a
previous study showing no increased risk of epilepsy
5 years after a mild brain injury.1
The discrepancy might
result from diﬀerent inclusion criteria for mild brain
injury and epilepsy,1,11
and an insuﬃcient sample size to
identify a moderate increase in risk.1
Our results suggest
that time from brain injury to clinically overt symptoms
(seizures) can span several years, leaving room for
clinical intervention.5
However, animal studies suggest
that a speciﬁc time window exists shortly after injury in
which appropriate drugs might stop the epileptogenic
process,22
and antiepileptogenic trials after brain injury
in human beings have not shown drug treatment to be
eﬀective.7
In Denmark, seizure prophylaxis with
antiepileptic drugs after brain injury was not used
routinely in the study period.23
We deﬁned the onset of epilepsy as the ﬁrst day of the
ﬁrst contact, although this is only an approximation.
There may be a delay from the ﬁrst seizure to diagnosis
of epilepsy. We have previously validated the epilepsy
diagnosis in a sample from the Danish National Hospital
Register and found that 64% were registered in the
Danish National Hospital Register within 1 year of ﬁrst
seizure, and 90% were registered within 5 years.11
Diagnostic delay might, therefore, explain part of the
increased risk of epilepsy after the brain injury. Likewise,
a delay between brain injury and diagnosis (eg, in
patients with chronic subdural haematoma), could bias
the estimates of epilepsy shortly after a brain injury
diagnosis, but this eﬀect is likely to be small, especially
in children.
Brain injury might be the ﬁrst presentation of epilepsy,
in which the patient has a head trauma during an
unwitnessed seizure (reverse causation). In a
subanalysis, we excluded patients diagnosed with
epilepsy within the ﬁrst 6 weeks of ﬁrst brain injury
diagnosis and found that the high risk of epilepsy
remained for all types of brain injury, albeit in an
attenuated form (data not shown). Although patients
with infrequent seizures might remain undiagnosed
more than 6 weeks, this problem probably aﬀects only a
small part of the delayed association between brain
injury and epilepsy. The risk of epilepsy increased
slightly with age at time of mild brain injury, and was
highest for people over 15 years of age, indicating that
susceptibility to epilepsy after brain injury increases
with age. This ﬁnding is in line with results of a previous
study identifying people aged 65 or more as being at
high risk of epilepsy after brain injury.1
Alternatively, the
severity of brain injuries might increase with age, or
doctors might be more likely to hospitalise younger
children with less severe brain injuries, resulting in a
Number of patients
with epilepsy
New cases (per
1000 person-years)
Adjusted relative risk#
(95% CI)
p value
Hospital stay (days) for mild brain injury
0 256 1·73 2·22 (1·96–2·51) <0·0001
1–6 563 1·60 2·20 (2·02–2·40) <0·0001
7–13 9 2·08 3·01 (1·56–5·78) 0·0010
14–27 4 2·54 3·68 (1·38–9·82) 0·0091
≥28 5 2·05 2·94 (1·22–7·07) 0·0159
No mild injury 16633 0·87 1·00 ··
Hospital stay (days) for severe brain injury
0 12 1·73 2·09 (1·19–3·68) <0·0108
1–6 24 3·71 4·82 (3·23–7·20) <0·0001
7–13 15 7·04 9·42 (5·68–15·63) <0·0001
14–27 18 13·11 18·01 (11·34–28·60) <0·0001
≥28 47 14·86 20·07 (15·06–26·74) <0·0001
No severe injury 17354 0·89 1·00 ··
Hospital stay (days) for skull fracture
0 8 2·13 2·72 (1·36–5·45) 0·0046
1–6 48 1·35 1·77 (1·33–2·35) <0·0001
7–13 10 1·99 2·70 (1·45–5·03) 0·0017
14–27 4 3·08 4·01 (1·51–10·69) 0·0055
≥28 8 5·59 6·69 (3·35–13·38) <0·0001
No fracture 17392 0·89 1·00 ··
Each form of injury led to a signiﬁcant (p<0·0001) increase in risk of epilepsy relative to people without brain injury.
Relative risk (RR) was adjusted for age and interaction with sex and calendar year. RR of epilepsy in people with brain
injury was modiﬁed by duration of ﬁrst hospital stay with brain injury for severe brain injury (p<0·0001) and skull
fracture (p=0·02) but not mild brain injury (p=0·73).
Table 3: Duration of ﬁrst hospital stay with brain injury and relative risk of epilepsy
No family history of epilepsy Family history of epilepsy
Number of
patients with
epilepsy
Adjusted relative
risk (95% CI)
p value Number of
patients with
epilepsy
Adjusted relative
risk (95% CI)
p value
Mild brain injury
No 15511 1·00 ·· 1122 3·37 (3·17–3·58) <0·0001
Yes 766 2·24 (2·08–2·41) <0·0001 71 5·75 (4·56–7·27) <0·0001
Severe brain injury
No 16166 1·00 ·· 1188 3·35 (3·16–3·56) <0·0001
Yes 11 7·81 (6·48–9·42) <0·0001 5 10·09 (4·20–24·26) <0·0001
Skull fracture
No 16202 1·00 ·· 1190 3·35 (3·16–3·55) <0·0001
Yes 75 2·28 (1·81–2·86) <0·0001 3 2·71 (0·87–8·41) 0·0842
Any brain injury
No 15338 1·00 ·· 1115 3·39 (3·19–3·61) <0·0001
Yes 939 2·47 (2·31–2·65) <0·0001 78 5·73 (4·58–7·16) <0·0001
Patients might have been exposed to more than one type of brain injury at separate admissions/outpatient visits.
Relative risk adjusted for age and its interaction with sex and calendar year.
Table 4: Family history and relative risk of epilepsy after traumatic brain injury

20.
Articles
www.thelancet.com Vol 373 March 28, 2009 1109
low relative risk of post-traumatic epilepsy in young age
groups.
Post-traumatic epilepsy is thought to be typical of
symptomatic epilepsy (ie, determined by environmental
factors). However, twin studies suggest that genetic
factors also play a part in localisation-related epilepsies,
most of which are thought to be symptomatic or
probably symptomatic.24
Family history of epilepsy and
mild brain injury independently contribute to the risk of
epilepsy.25
Thus, people genetically predisposed to
epilepsy (ie, with a family history of epilepsy) have a
higher risk of epilepsy than do people without genetic
predisposition when exposed to mild brain injury. To
our knowledge, no previous studies have studied the
risk of epilepsy after brain injury in ﬁrst degree relatives
to patients with epilepsy. In animals, variation in the
susceptibility of various rat strains to post-traumatic
epilepsy might lend some support to the hypothesis of an
underlying genetically determined tendency to develop
post-traumatic epilepsy.26
Our registration of family history is not complete
because some parents and older siblings might have
been diagnosed before the Danish National Hospital
Register was established (Jan 1, 1977). This mis-
classiﬁcation is likely to cause an underestimation of the
eﬀect of family history on the risk of epilepsy.
The relative risk of epilepsy after mild brain injury was
slightly higher in female than in male patients perhaps
because female patients with epilepsy are more likely
registered in the National Hospital Register because of
sex-speciﬁc factors, such as pregnancy. Alternatively
female brains might be more susceptible to epilepsy after
mild brain injury than are male brain, as supported by a
previous study showing that localisation-related epilepsy
with no apparent structural cause is more prevalent in
women than in men.27
The sex diﬀerence was not present
for the other types of brain injury, suggesting that other
mechanisms might be involved in post-traumatic epilepsy
after skull fracture and more severe brain injuries.
The length of ﬁrst hospital stay with brain injury was
associated with an increased risk of epilepsy for severe
brain injury and cranial fractures. The length of
admission is probably related to severity of brain injury.
Despite the length and completeness of follow up, the
size of the study cohort, and the population-based nature
of the study,8
we had limited clinical information. In a
recent study, we validated the epilepsy diagnosis in the
Danish National Hospital Register.11
We found a positive
predictive value of an ICD-8 or ICD-10 epilepsy diagnosis
according to ILAE criteria28
of 81% for epilepsy and
89% for single seizures, but identiﬁed no epilepsy
diagnoses based on acute symptomatic seizures.11
Thus,
some patients registered with epilepsy in the present
study do not fulﬁl the diagnostic criteria, but the
misclassiﬁcation would only bias the results of the
present study away from the null hypothesis if the
quality of the epilepsy registration diﬀers between
patients with and without brain injury, which we ﬁnd
unlikely.
The Danish Hospital Register does not capture all
patients with epilepsy, because some outpatients might
be treated in private practice. However, estimates of
incidence (68·8 per 100000 people per year) and
prevalence (0·6%) of epilepsy in Denmark based on
data from the Danish National Hospital Register were
similar to those found in other developed countries and
indicate a high completeness.10
If cases with epilepsy are
missed in the Danish National Hospital Register, the
relative risk of epilepsy would be aﬀected only if the
incomplete capture of patients diﬀers between those
with and without brain injury. Patients with head injury
may be followed more closely than the general
population, which might increase the completeness and
overestimate the relative risk of epilepsy after brain
injury. However, the eﬀect of this bias is likely to
decrease over time.
Although, most patients with epilepsy are cared for on
an outpatient basis, the incidence estimate only increased
by 17% after inclusion of outpatients.10
Hence, most
outpatients with epilepsy are also admitted to hospital
for that or other reasons and, thereby, included in the
National Hospital Register. Some patients with severe
brain injury live in care homes in the community after
their condition has stabilised, but these patients have the
same access to the hospital system as patients without
brain injury, and thus we think that they do not have a
decreased likelihood of being registered with epilepsy in
the Danish National Hospital Register.
People were censored when they died or left Denmark
permanently, but less than 3% of the entire cohort did
so.8
Some people may have had a brain injury or epilepsy
during a short stay abroad; but numbers are likely to be
very low, and most of these will be treated in Danish
hospitals or outpatient clinics when they return to
Denmark. Bias due to selection of study participants is
therefore an unlikely explanation for our ﬁndings. In
comparison, 1139 (25%) patients of a total population of
4541 in the Rochester study were lost to follow-up due to
migration from Minnesota.1
A previous study assessed the validity of the hospital
codes for brain injury (ICD-8: 851–854) showing that the
diagnoseswereconﬁrmedinabout88%ofcases.29
However,
clinical discrimination between diﬀerent types of brain
injury is diﬃcult and the deﬁnitions vary between
countries.14
Brain injuries that at ﬁrst seem mild can turn
outtobesevere.Inastudyof24patientswithpost-traumatic
amnesia lasting more than 1 week, four had initially been
diagnosed with skull fracture and four with concussion.14
Although, there is debate about the importance of
post-traumatic amnesia in the diagnosis of patients with
mild brain injury,14,30
some patients diagnosed with mild
head injury might actually suﬀer from more severe brain
injury, which would likely lead to an overestimated risk of
epilepsy associated with mild brain injury.

22.
Comment
1060 www.thelancet.com Vol 373 March 28, 2009
be able to provide a deﬁnitive treatment decision.9
To
reﬁne the indication for adjuvant treatment remains the
big task for futures studies.
Peter Hohenberger
Division of Surgical Oncology andThoracic Surgery,
Department of Surgery, Medical Faculty Mannheim,
University of Heidelberg, D-68135 Mannheim, Germany
peter.hohenberger@chir.ma.uni-heidelberg.de
I have received research grants and honoraria from Novartis.
1 Casali PG, Jost L, Reichardt P, Schlemmer M, Blay J-Y, on behalf of the ESMO
GuidelinesWorking Group. Gastrointestinal stromal tumors: ESMO clinical
recommendations for diagnosis, treatment and follow-up. Ann Oncol
2008; 19 (suppl 2): ii35–38.
2 DeMatteo RP, Ballman KV, Antonescu CR, on behalf of the American
College of Surgeons Oncology Group (ACOSOG) Intergroup Adjuvant GIST
StudyTeam. Adjuvant imatinib mesylate after resection of localised,
primary gastrointestinal stromal tumour: a randomised, double-blind,
placebo-controlled trial. Lancet 2009; published online March 19.
DOI:10.1016/S0140-6736(09)60500-6.
3 Verweij J, Casali PG, Zalcberg J, et al. Progression-free survival in
gastrointestinal stromal tumours with high-dose imatinib: randomised
trial. Lancet 2004; 364: 1127–34.
4 Debiec-Rychter M, Sciot R, Le Cesne A, et al, on behalf of the EORTC Soft
Tissue and Bone Sarcoma Group,The Italian Sarcoma Group and the
Australasian GastroIntestinalTrials Group. KIT mutations and dose
selection for imatinib in patients with advanced gastrointestinal stromal
tumours. Eur J Cancer 2006; 42: 1093–103.
5 Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathology and
prognosis at diﬀerent sites. Semin Diagn Pathol 2006; 23: 70–83.
6 Corless CL, Schroeder A, Griﬃth D, et al. PDGFRA mutations in
gastrointestinal stromal tumors: frequency, spectrum and in vitro
sensitivity to imatinib. J Clin Oncol 2005; 23: 5357–64.
7 Mussi C, Schildhaus HU, Gronchi A, Wardelmann E, Hohenberger P.
Therapeutic consequences from molecular biology for GIST patients
aﬀected by neuroﬁbromatosis type 1. Clin Cancer Res 2008; 14: 4550–55.
8 Fletcher CD, Berman JJ, Corless C, et al. Diagnosis of gastrointestinal
stromal tumors: a consensus approach. Hum Pathol 2002; 33: 459–65.
9 GronchiA, Judson I, NishidaT, et al.Adjuvanttreatment ofGIST with
imatinib: solid ground or still quicksand?A comment on behalf ofthe EORTC
SoftTissue and Bone SarcomaGroup,the Italian SarcomaGroup,the NCRI
SarcomaClinical StudiesGroup (UK),the Japanese StudyGroup onGIST,the
French SarcomaGroup andthe Spanish SarcomaGroup (GEIS). Eur JCancer
2009;published online March 16. DOI:10.1016/j.ejca.2009.02.009.
Risk of epilepsy after head trauma
Head trauma is an important cause of epilepsy, and
knowledge of the extent of the risk of epilepsy after
head trauma and the factors that inﬂuence this risk
are essential. In The Lancet today, Jakob Christensen
and colleagues1
present their population-based cohort
study of more than 1·5 million people born in Denmark
between 1977 and 2002, and followed up for that
period. 78572 of them had at least one head injury
and 17470 were diagnosed with epilepsy, of whom
1017 had had a head injury before diagnosis. These
researchers obtained the data from the Danish National
Hospital Register, which provided diagnostic coding on
inpatients from 1977, and outpatients from 1995, on
epilepsy and head injury. Family history was ascertained
by linkage of data from ﬁrst-degree relatives. The
researchers compared the relative risks of development
of epilepsy for people with mild and severe head injury
(with or without a family history of epilepsy) on a yearly
basis with those for people without head injury, while
controlling for age, sex, and calendar year.
Overall, the relative risks of epilepsy were raised about
two-fold (relative risk 2·2) after a mild head injury and
seven-fold (7·4) after a severe head injury, were slightly
greater in women than in men, and increased with
older age at time of injury. The rate of development of
epilepsy was greatest in the few years after the head
injury; for instance,with a greaterthan ﬁve-fold increase
for 2–3 years after a severe head injury, but the excess
risk continued for 10 years after mild and severe brain
injury—longer than in other studies.2
The incidence
of epilepsy was greater in head-injured people with a
family history of epilepsy than in those without a family
history, with about a six-fold increase in the relative
risk of epilepsy after a mild head injury and a ten-fold
increase after a severe injury. This ﬁnding emphasises
that the cause of epilepsy is often multifactorial.
Previous studies in this area have been either too
small or open to too many methodological criticisms to
be deemed to provide deﬁnitive data. Christensen and
co-workers’ investigation is of commendable size and
completeness, with an advanced statistical design—as
such, it should be accepted as the reference study in the
ﬁeld.This is not to say that there are not methodological
criticisms. There are issues inherent in the study design:
the diagnosis of epilepsy and the classiﬁcation of
severity of trauma are based on registry data, with all
the inaccuracy that this implies; no attempt is made
to distinguish between immediate, early, and late
epilepsy although these categories have important
clinical implications; previously identiﬁed risk factors
for post-traumatic epilepsy, such as the presence of
dural tear, intracranial haemorrhage, and early seizures
(<1 week) were not investigated; and no data are
provided about the type or severity of the epilepsy.
Published Online
February 23, 2009
DOI:10.1016/S0140-
6736(09)60215-4
See Articles page 1105

23.
Comment
www.thelancet.com Vol 373 March 28, 2009 1061
The decision about whether or not to give anti-
epileptic drugs prophylactically in patients with head
injury is a common clinical dilemma. Christensen
and co-workers’ study does not address the value of
treatment, but the risk estimates will help patients
and doctors make decisions more clearly. The study will
also be of value in helping to determine epilepsy risks
for medicolegal purposes, by providing a sound basis
for determination of cause and compensation and, as
such, is a service to social justice. Scientiﬁc value exists
too in the ﬁnding that the risk of epilepsy was increased
for at least 10 years after head injury. Post-traumatic
epileptogenesis is thus a long process, which raises
the possibility that neuroprotective measures3
could
interfere with this process and thus reduce the risk of
epilepsy. Past attempts to prevent epilepsy have been
disappointing,4
but these new data suggest that such
eﬀorts should be renewed, to focus particularly on
high-risk groups (those with severe head injury, within
2 years of injury, and a positive family history).
Finally, we should note the value of such large-scale
epidemiological studiesthatuse pre-existingdatabases.
Such studies are increasingly diﬃcult to do in the UK,
for example, because of sometimes over-zealous inter-
pretation of conﬁdentiality and consent regulations,
and the timidity of the bureaucratic processes. In the
UK, we have reached a situation in which, in large
swathes of clinical epidemiological research, the baby
is being well and truly thrown out with the bathwater,
to the detriment of patients and the acquisition of
beneﬁcial knowledge.5
*Simon Shorvon, Aidan Neligan
University College London Institute of Neurology, National
Hospital for Neurology and Neurosurgery, LondonWC1N 3BG, UK
s.shorvon@ion.ucl.ac.uk
We declare that we have no conﬂict of interest.
1 Christensen JC, Pedersen MG, Pedersen CB, Sidenius P, Olsen J,
Vestergaard M. Long-term risk of epilepsy after traumatic brain injury in
children and young adults: a population-based cohort study. Lancet
2009; published online Feb 23. DOI:10.1016/S0140-6736(09)60214-2.
2 Annegers JF, Hauser WA, Coan SP, Rocca WA. A population-based
study of seizures after traumatic brain injuries. N Engl J Med 1998;
338: 20–24.
3 Temkin NR. Antiepileptogenesis and seizure prevention trials with
antiepileptic drugs: meta-analysis of controlled trials. Epilepsia 2001;
42: 515–24.
4 Temkin NR, Dikmen SS,Wilensky AJ, Keihm J, Chabal S,Winn HR.
A randomized, double-blind study of phenytoin for the prevention of
post-traumatic seizures. N Engl J Med 1990; 323: 497–502.
5 Metcalfe C, Martin RM, Noble S, et al. Low risk research using routinely
collected identiﬁable health information without informed consent:
encounters with the Patient Information Advisory Group. J Med Ethics
2008; 34: 37–40.
Subdural haematoma (red) in 10-year-old boy following trauma
SciencePhotoLibrary
Elimination of blinding trachoma revolves around children
Blinding trachoma is a terrible disease. The intense
conjunctival inﬂammation in young children causes
conjunctival scarring, leading in adult life to inturned
eyelashes (trichiasis) that rub on the eye and cause
painful blindness. In The Lancet today, Jenaﬁr House and
colleagues report a study in hyperendemic communities
in Ethiopia.1
In such areas more than half of children are
aﬀected, almost every adult has scarring, and 10–20% of
older people have trichiasis. Trachoma is now restricted
to poor developing areas, having disappeared from
Europe and North America where only a century ago it
was a major problem.
Chlamydia trachomatis, the causative bacterium
for trachoma, has evolved with human beings and
their vertebrate ancestors since Jurassic times.2
It has
developed an eﬀective host–parasite relation over a
See Articles page 1111

25.
physiological processes in brain development, including the
proliferation, migration, survival and differentiation of neurons,
blockade of excessive NMDA receptor activity must be achieved
without affecting normal brain functioning (Kohr, 2007).
Recently, increasing evidence based on molecular studies
suggests that memantine, an uncompetitive NMDA receptor
blocker with fast channel unblocking kinetics to prevent it from
occupying the channels and interfering with normal synaptic
transmission, is a potent neuroprotectant without above-
mentioned side effects (Chen et al., 1992, 1998; Chen and Lipton,
2005; Johnson and Kotermanski, 2006). In contrast to MK-801
and ketamine, memantine shows unusual clinical tolerance in
the treatment of moderate-to-severe Alzheimer's disease in
adults through its low affinity and relatively fast unblocking
kinetics (de Lima et al., 2000; Lipton, 2004; Lipton, 2006). As a
neuroprotective agent, memantine can reduce functional as
well as morphological sequelae induced by ischemia (Block and
Schwarz, 1996; Chen et al., 1998). A recent study showed the
NMDA receptor blockade with memantine could provide an
effective pharmacological prevention of periventricular leuko-
malacia (PVL) in the premature infant (Manning et al., 2008).
Topiramate, a well tolerated antiepileptic drug (AED) used
clinically, confers neuroprotection by blocking AMPA/KA
receptors and use-dependent Na+
channel in developing rat
brain without serious side effects compared to conventional
anticonvulsants (Noh et al., 2006). Topiramate has anti-
excitotoxic properties, because it protects against motor
neuron degeneration. The other neuroprotective effects of
topiramate include positive modulation of gamma-aminobu-
tyric acid (GABA) receptors, increase of seizure threshold and
so on (Pappalardo et al., 2004). Furthermore, Topiramate also
protects preoligodendrocytes against excitotoxic cellular
death in white matter lesions and prevents the periventricular
white matter from the damage induced by an AMPA/KA
agonist in newborn mice (Follett et al., 2004; Sfaello et al., 2005).
Due to the complex pathological mechanisms in HIBI
described above, combination therapy or multimodal target-
ing is thought to be a key future approach to provide effective
neuroprotection. Most promising combination should target
different neuroprotective mechanisms, expand the therapeu-
tic time window, and alleviate the possibility of side effects
(Rogalewski et al., 2006). Studies on the mechanisms of the
superfamily of glutamate receptors revealed that NMDA and
AMPA glutamate receptors showed a fine-tuned interaction at
the glutamatergic synapse: the rapid activation and brief open
time of AMPA receptors facilitates unblock of NMDA receptors
(Villmann and Becker, 2007). Functional interdependence of
AMPA and NMDA receptors has been proven by experiments
where a transient synaptic activation of NMDA receptors
reliably induces a long-term potentiation phenomenon,
associated with an increase in the intensity and number of
synaptic AMPA-receptor clusters (Liao et al., 2001; Liu et al.,
2004a). These findings suggest that it will be more effective
and beneficial to block both NMDA and AMPA/KA receptors by
combination of different glutamate receptor antagonists.
Based on the pharmacology and mechanism studies, we
designed the experiments to evaluate the efficacy of the
combination therapy by measuring gross brain damage, brain
weight deficit in the right hemisphere and regional neuronal
injury. Besides the morphologic and histopathologic measure-
ment, a neurofunctional test was performed to verify the
results. To ensure therapeutic safety, the possible drug-
induced apoptosis was assessed even though the two drugs
were approved safe and efficient in their respective therapeu-
tic categories (Chen et al., 1998; Glier et al., 2004).
2. Results
2.1. Gross brain damage grading
The neurologic damage score was determined by an observer
blind to the drug treatment of the rat pups. Table 1 shows the
neurologic damage scores in each group. The neurologic
damage score was significantly higher in the vehicle-treated
group (2.79±1.23, n=19) than that in the combination-treated
Table 1 – Neurologic damage score.
Group No. Normal=1 Mild=2 Moderate=3 Severe=4 p ⁎
Vehicle 19 4 4 3 8 NS
Memantine 24 10 7 4 3 <0.05
Topiramate 21 5 5 5 6 >0.05
Combination 24 13 6 4 1 <0.01
The number of pups receiving the designated gross damage score by a blinded observer.
⁎ p value, memantine, topiramate, combination vs. vehicle.
Fig. 1 – The percentage of reduction in right cerebral
hemisphere weight measured using the left hemisphere
weight as standard. The animal numbers are as described in
the result. The percentage of reduction in right hemisphere
weight was significantly decreased in the combination group
compared with the vehicle group (**p<0.01 vs. vehicle). The
percentage of reduction in right hemisphere weight was
significantly decreased in the memantine group compared
with the vehicle group (*p<0.05 vs. vehicle). Data are
presented as mean±S.D.
174 B R A I N R E S E A R C H 1 2 8 2 ( 2 0 0 9 ) 1 7 3 – 1 8 2

26.
group (1.71±0.91, n=24, p<0.01 versus vehicle). The neurologic
damage score was significantly higher in the vehicle-treated
group than that in the memantine-treated group (2.00±1.06,
n=24, p<0.05 versus vehicle). The neurologic damage score in
the topiramate-treated group (2.57±1.17, n=21, p>0.05 versus
vehicle) was lower but not statistically significant compared
with the vehicle-treated group.
2.2. Brain weight deficit
Fig. 1 shows the weight deficit in the right hemisphere relative
to the left hemisphere. The weight deficit in the combination-
treated group (9.2±2.5%, n=24, p<0.01 versus vehicle) was
significantly reduced compared with the vehicle-treated
group (26.9±4.1%, n=19). The weight deficit in the meman-
tine-treated group (16.3±3.2%, n=24, p<0.05 versus vehicle)
was significantly reduced compared with the vehicle-treated
group. The weight deficit in the topiramate-treated group
(21.5±4.0%, n=21, p>0.05 versus vehicle) was reduced but
not statistically significant compared with the vehicle-
treated group. Body weights of rat pups in each group were
recorded and analyzed. Results showed that the body
weights of the treated groups were not significantly different
from the vehicle-treated group at 1, 3, 7, 14 and 22 days after
injury (data not shown). Mortality rates were not signifi-
cantly different in four groups, although there was a trend
toward reduced mortality in the combination group.
2.3. Microscopic brain damage grading
The microscopic brain damage score(histopathologic score) was
determined by an observer blind to the drug treatment of the rat
pups. Fig. 2 shows the microscopic brain damage score in each
group. The histopathologic score in the memantine-treated
group (2.15±0.52 and 1.51±0.47, n=12, p<0.05 and p<0.05 versus
vehicle) was significantly lower compared with the vehicle-
treated group (4.15±0.73 and 3.38±0.72, n=10) in the cortex and
thalamus. The histopathologic score in the combination-treated
group (1.91±0.51, 1.45±0.49 and 0.91±0.42, n=12, p<0.05,
p<0.05 and p<0.01 versus vehicle) was significantly lower
compared with the vehicle-treated group (4.15±0.73, 3.68±0.62
and 3.38±0.72, n=10) in the cortex, hippocampus and thalamus.
In the striatum, the histopathologic score in the combination-
treated group was lower but not statistically significant
compared with the vehicle-treated group.
2.4. Foot-fault test
Fig. 3 shows the number of foot-faults in each group. The
number of foot-faults per pup was significantly greater in the
vehicle-treated group (8.62±1.51, n=10) than that in the
Fig. 2 – Microscopic brain damage scores in the cortex, hippocampus, striatum, thalamus. Data are presented as mean±S.D.
*p<0.05 vs. vehicle, **p<0.01 vs. vehicle.
Fig. 3 – Number of foot-faults in each group. The combination
group had significantly fewer foot-faults than the vehicle
group. Data are presented as mean±S.D. *p<0.05 vs. vehicle.
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27.
combination-treated group (4.26±0.93, n=12, p<0.05 versus
vehicle). The number of foot-faults per pup was significantly
greater in the vehicle-treated group than that in the meman-
tine-treated group (4.66±1.03, n=12, p<0.05 versus vehicle).
The number of foot-faults per pup was less but not statistically
significant in the topiramate-treated group (6.94±1.22, n=11)
compared with in the vehicle-treated group.
2.5. TUNEL-positive cell counting
The numbers of TUNEL-positive apoptotic cells of each group
are presented in Fig. 4 and areas examined for drug-induced
apoptosis are shown in Fig. 5. In all observed areas, the
numbers of apoptotic cells in the treated group (single or
combined) were not significantly increased compared with the
vehicle-treated group. In the CA1 sector of the hippocampus,
The numbers of apoptotic cells in the combination-treated
group (31.2±20.7 and 45.5±31.2, n=12, p<0.01 and p<0.01
versus vehicle) were significantly reduced compared with the
vehicle-treated group (82.1±32.6 and 175±48.2, n=12). In the
CA1 sector of the hippocampus and the subcortical white
matter, The numbers of apoptotic cells in the memantine-
treated group (50.5±28.3 and 99.8±38.7, n=12, p<0.05 and
p<0.05 versus vehicle) were significantly reduced compared
with the vehicle-treated group. In other areas, no significant
differences were found between any of the treated groups
(single or combined) and the vehicle group. Fig. 6 shows some
sample pictures of apoptotic cells in the CA1 sector of the
hippocampus.
3. Discussion
The present study shows for the first time to our knowledge
that the combination of memantine and topiramate exerts
enhanced protection of neurons against HIBI in vivo, compared
with each of these agents alone. In this study, we measured
brain damage in each group by using the gross anatomic
method of Palmer et al. at 22d post-HI. By delaying assessment
until 22d after HI, we included very late cell death that reflects
overall neuroprotective effect of the drugs in a relatively long
period. We also examined the brain weight deficit presented
by the loss of brain weight on the ipsilateral side relative to the
contralateral side. Results showed the combination therapy
significantly reduced the degree of brain injury in this model.
Besides the morphologic examinations, we applied the foot-
Fig. 4 – The numbers of TUNEL-positive apoptotic cells in the cortex, the CA1, CA3 and dentate gyrus of the hippocampus, the
striatum and the subcortical white matter in the vehicle, memantine, topiramate and combination group. Data are presented as
mean±S.D. *p<0.05 vs. vehicle, **p<0.01 vs. vehicle.
Fig. 5 – Areas of the brain examined for neuronal injury and
drug-induced apoptosis. CX=cortex CA1=hippocampus CA1
CA3=hippocampus CA3 Den=dentate gyrus ST=striatum
TH=thalamus.
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28.
fault test to evaluate sensorimotor function of the rat pups at
21d post-HI. Foot-faults per pup in the combination group
were significantly less than that in the vehicle group. The
functional outcome was consistent with the morphologic
findings in the long-term perspective. The short-term effect of
the combination therapy was evaluated by microscopic brain
damage scoring at 72 h post-HI. Results showed that the
combination therapy reduced neuronal injury significantly in
the cortex, hippocampus and thalamus.
Neuronal cell death after HI has generally been attributed
to either rapid necrosis or delayed apoptosis. There is no doubt
that necrosis plays major role in the course. But the develop-
ing brain may have good plasticity and a high capacity for self-
repair (Daval et al., 2004; Grafe, 1994). After most compensa-
tory and reparative phrases have passed, there are at least
three different end points should be taken into account in
assessment: the long-term deficit of brain tissue, the func-
tional consequences of the brain injury and the acute extent of
brain injury (Bona et al., 1997). The quantitive assessment of
brain weight deficit and gross brain damage used in this study
can accurately evaluate neuroprotective effects of glutamate
antagonists against NMDA-mediated brain injury in vivo
(Andine et al., 1990; McDonald et al., 1989a). On the other
hand, behavioral consequences after HIBI are essential to
reveal the true functional disability and to study the effects of
drug intervention. In this study, the foot-fault test was done at
21d post-HI to evaluate the long-term functional outcome.
Different from other cognitive function tests (Morris water
maze, etc) related mostly to the hippocampus formation, the
foot-fault test correlates with brain lesion in the cerebral
cortex which is the most constantly affected region in both
mild and severe HIBI in this model (Bona et al., 1997). Short-
term effect of the therapy was evaluated by a scoring system
on neuronal injury in 4 main regions of the rat brain at 72 h
post-HI. Because short-term neuronal injury in the developing
brain after HI is caused by both early and delayed neurode-
generation, the onset of damage in different regions of the
brain is time-dependent and progressive, and it has an uneven
distribution within regions (Northington et al., 2001). However,
72h (3d) post-HI seems an appropriate time point to evaluate
short-term neuronal injury after insult in this model (Feng et
al., 2005, 2008; Manning et al., 2008; Zhu et al., 2004).
In our experiment, the time window and doses of
memantine and topiramate were chosen according to a
general purpose to achieve an application for potential clinical
use. Based on published data of rat pharmacokinetics and
dose–response studies, 20 mg/kg dose of memantine can
provide minimal neuroprotection (Chen et al., 1998; Hesselink
et al., 1999). Considering the short therapeutic time window
(Culmsee et al., 2004) and the confirmed neuroprotective
effects of memantine at 20 mg/kg dose in HI and PVL model,
we administered the 20 mg/kg loading dose of memantine
immediately after HI in the treatment. Topiramate (loading
dose 50 mg/kg, maintenance dose 20 mg/kg/day) can reduce
neuronal cell loss significantly but increase apoptosis in the
frontal white matter in newborn piglets (Schubert et al., 2005).
Furthermore, topiramate may cause neurodegeneration in the
developing rat brain only at doses at and above 50 mg/kg
(Glier et al., 2004). The reason why topiramate at doses above
50 mg/kg can protect neurons but increase apoptosis may
relate to two mechanisms. The first one is the blockade of
AMPA/KA receptors lack of interference with NMDA-receptor
signaling (Gibbs et al., 2000). Topiramate cannot provide
neuroprotection only through AMPA/KA receptor channel
unless it reaches threshold dosage. The second one is the
depression of the endogenous neurotrophin system in the
brain which may account for the proapoptotic effect (Bittigau
et al., 2002). In a gerbil model, topiramate was found reducing
Fig. 6 – Sample pictures of TUNEL-positive apoptotic cells in the CA1 sector of the hippocampus in the (A) vehicle,
(B) memantine, (C) topiramate and (D) combination group. Original magnification, ×400.
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29.
hippocampal neuronal damage in dose-dependent manner
(Lee et al., 2000). Based on the dose–response studies and our
preliminary experiment, we chose 40 mg/kg as the loading
dose for topiramate. The dose of topiramate (loading dose
40 mg/kg; maintenance dose 20 mg/kg/day) was proven
considerably safe but unlikely to be neuroprotective.
Although the mechanisms underlying the neuroprotection
are not fully understood, the results demonstrate that a
synergistic reduction in brain damage can be achieved
effectively by memantine combined with topiramate. The
neuroprotective actions and unique characteristics of these
two drugs may account for the experimental outcome. It is
well documented that memantine antagonizes NMDA recep-
tor activation by inhibiting the influx of Ca2+
through this
channel (Johnson and Kotermanski, 2006). As an open-
channel blocker, memantine can provide neuroprotection
without interference with the normal brain development
(Parsons et al., 1999). The favorable kinetics of memantine
interaction with NMDA channels may be partly responsible for
its high index of therapeutic safety, and it makes memantine a
candidate drug for use in many NMDA receptor-mediated
human CNS disorders (Johnson and Kotermanski, 2006;
Lipton, 2004). In a four-vessel-occlusion (4VO) global ischemic
model, neuronal damage in the CA1 sector of the hippocam-
pus and in the striatum produced by 4VO was significantly
attenuated by 20 mg/kg memantine (Block and Schwarz, 1996).
Memantine has been used clinically for excitotoxic disorders
at neuroprotective doses administered up to 2 h after
induction of HI in immature and adult rats. At neuroprotective
concentrations, memantine results in few adverse side effects
and displays virtually no effects on Morris water maze
performance or on neuronal vacuolation (Chen et al., 1998).
Rosi et al. found that memantine protects against LPS-induced
neuroinflammation, and confers neural and cognitive protec-
tion (Rosi et al., 2006). Furthermore, NMDA receptor blockade
with memantine can provide an effective pharmacological
prevention of PVL in the premature infant without affecting
normal myelination or cortical growth (Manning et al., 2008).
Topiramate is a novel broad spectrum antiepileptic drug
(AED) used clinically in adults and children older than 2 years.
Amongst new-generation AEDs examined for neurotoxicity in
neonatal rats, topiramate holds promise for minimizing the
risk of neuronal death without side effects such as the
impairment of cognitive performance (Cha et al., 2002; Glier
et al., 2004; Mellon et al., 2007). Pharmacological actions of
topiramate include positive modulation of GABA receptors,
inhibition of the AMPA/KA glutamate receptor subtypes and
blockade of a use-dependent Na+ channel (Schubert et al.,
2005). Noh and his coworkers reported the co-treatment of
topiramate and an NMDA receptor antagonist D-AP5 greatly
increased the number of viable neurons in oxygen–glucose
deprivated cells. The experiment determined that neuropro-
tective effect of topiramate was mainly mediated by the
inhibition of AMPA glutamate receptors (Noh et al., 2006).
Topiramate blocks the spread of seizures caused by transient
global cerebral ischemia, and reduces the abnormally high
extracellular levels of glutamate in the hippocampus in the
immature rat spontaneous epileptic model by blocking AMPA
receptors (Koh et al., 2004). It also affects the expression of
glutamate transporters (GLAST and GLT-1) which are respon-
sible for the inactivation of glutamate as a neurotransmitter
(Poulsen et al., 2006). Moreover, topiramate was found
effective in attenuating seizure-induced neuronal cell death
and reducing KA-induced Phospho-extracellular signal-regu-
lated kinase-immunoreactive (p-Erk IR) in the CA3 region of
the hippocampus (Park et al., 2008). In a rat pup model of PVL,
topiramate has been demonstrated effective to attenuate
AMPA/KA receptor-mediated cell death and Ca2+
influx, as
well as KA-evoked currents in developing oligodendrocytes
(Follett et al., 2004).
Many studies suggest that combination of drugs may
produce greater toxicity than individual ones. Thus, the safety
of combination therapies should be most concerned, when
these animal findings are intended for extrapolating to a
pediatric surgical patient population (Bittigau et al., 2002). The
rat is most sensitive to NMDA receptor-mediated neurotoxi-
city during early neuronal pathway development, referred to
as the “brain-growth spurt period” or period of synaptogen-
esis. (Haberny et al., 2002). Blockade of NMDA receptors up to
4 h is sufficient to trigger apoptotic neurodegeneration in the
developing brain (Ikonomidou et al., 1999). In consideration of
the possible neurotoxicity caused by the coadminstration of
drugs and the complicated interaction between NMDA recep-
tor blocker and AMPA receptor blocker, we examined the
possible drug-induced neuronal apoptosis by TUNEL staining
at 48 h post-HI even through the two drugs are proven safe at
the given doses respectively (Chen et al., 1998; Glier et al.,
2004). The time course of apoptotic injury varies regionally
because HI damage generally evolves more rapidly in the
immature brain than its adult counterpart. Injury in the cortex
and striatum occurs in a biphasic manner, where the early
phase (by 3 h) is classified as necrosis and the later phase (by
48 h) displays signs of apoptosis (Northington et al., 2001).
Nakajima et al. found that the density of caspase-3 immunor-
eactivity was enhanced in the frontal, parietal, and cingulate
cortex and in the striatum 24 h after hypoxic ischemic injury.
In the CA3 sector of the hippocampus, the dentate gyrus,
medial habenula and laterodorsal thalamus, the density of
apoptotic cells was highest at 24–72 h after HI and then
declined. In thalamus, increased caspase-3 immunoreactivity
was distributed in lateral, laterodorsal, and reticular nuclei
with a peak in density at 48 h after HI. In hippocampus,
intense caspase-3 immunoreactivity was present in CA1 and
in the dentate gyrus at 48 h after insult but had nearly
disappeared by 7d after HI injury (Nakajima et al., 2000).
Based on all these results on apoptotic injury, the time point
(48 h post-HI) was chosen to examine the apoptotic
neurodegeneration.
In this experiment, massive cellular apoptosis was not
found in all observed areas in the treated groups, and
apoptosis was reduced in the CA1 sector of the hippocampus
and the subcortical white matter in the combination group
compared with the vehicle group. The safe dosing regimen
and anti-apoptotic actions of memantine and topiramate may
contribute to the results synergistically. Regional patterns of
neuronal death can also be detected by expression of caspase-
3, a cysteine protease involved in the execution phase of
apoptosis. Immunocytochemical and Western blot analyses
show increased caspase-3 expression in damaged hemi-
spheres 24 h to 7d after HI. Reduced caspase-3 activity has
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30.
been shown to be associated with neuroprotection (Endres et
al., 1998; Puka-Sundvall et al., 2000). Memantine (20 mg/kg, i.
p.) can prevent isoflurane-induced caspase-3 activation and
apoptosis in vivo and in vitro. The results also indicated that
isoflurane-induced caspase activation and apoptosis are
dependent on cytosolic calcium levels (Zhang et al., 2008). In
recent years, many studies focus on the protection of white
matter because the importance of PVL pathophysiology has
been realized gradually (Khwaja and Volpe, 2008; Volpe, 2008).
NMDA receptor blockade with memantine acts as an effective
pharmacological contributor with little side effects in attenu-
ating white matter injury, and the protective dose of
memantine does not affect normal myelination or cortical
growth (Manning et al., 2008; Micu et al., 2006). In our
experiment, the apoptosis in the subcortical white matter
was reduced significantly in the combination group, which is
consistent with the previous findings on caspase-3 activation.
The present study demonstrated that a synergistic reduc-
tion in brain damage could be achieved by combination of
neuroprotective agents targeting different mechanisms.
Although an evolving body of work has shown that combina-
tion therapy holds promise in the treatment of HIBI, there has
been relatively little research on the combination therapy of
two glutamate receptor antagonists. The combination of
NMDA receptor antagonist MK-801 and AMPA receptor
antagonist NBQX shows an “overadditive” effect in cell culture
and focal ischemia model in mice (Lippert et al., 1994). On the
other hand, several studies on memantine or topiramate have
shown multidrug strategies are required for optimal thera-
peutic outcome. The combination of memantine and clenbu-
terol not only reduces the infarct size but also extends the
therapeutic window of clenbuterol up to 2 h after ischemia
(Culmsee et al., 2004). The combination of memantine and
celecoxib shows better effects in neuroprotection and anti-
inflammation in intracerebral hemorrhage treatment (Sinn et
al., 2007). Combined treatment with topiramate and delayed
hypothermia improves both performance and pathological
outcome in P15 and P35 rats (Liu et al., 2004b).
Although the present study demonstrates the neuroprotec-
tive effect of memantine combined with topiramate, further
studies are still needed in two aspects. A full dose–response
experiment was not performed in the present study, so further
investigation is still needed to determine the most optimal
dosing regimen of memantine and topiramate. Noh et al.
suggested that the pretreatment with topiramate before HI
was more effective than the post-treatment after HI (Noh et al.,
2006). The result implies that the pretreatment with topiramate
in the combination therapy can be considered in the future.
Collectively, the present study not only shows a promising
therapy for neuroprotection, but also proposes a new para-
digm for multidrug development which is thought to be a
promising approach in the treatment of HIBI.
4. Experimental procedures
4.1. Animal procedures
Seven-day-old rat pups of either sex, weighing between 12 g
and 16 g, were used in this study. The rat pups were randomly
assigned to one of the following groups: vehicle group (saline),
memantine group, topiramate group, combination group
(memantine and topiramate). All animal experiments fol-
lowed a protocol approved by the ethical committee on animal
research at our institution. The neonatal HI brain damage was
induced according to the modified Levine–Rice procedure
(Northington, 2006; Rice et al., 1981; Vannucci and Vannucci,
2005). For short, rat pups were anaesthetized by halothane
inhalation and duration of anesthesia was less than 5 min.
The right common carotid artery was dissected, and doubly
ligated. One hour later, rats were then placed in a plastic
chamber (37 °C) and exposed to 8% oxygen and 92% nitrogen
for 2 h. After this hypoxic exposure, the pups were returned to
their dams for 2 h recovery.
4.2. Drug administration
During recovery from HI, drugs were injected intraperitone-
ally: vehicle group received vehicle (0.5 ml 0.9% saline)
immediately after HI; memantine group received 20 mg/kg
loading dose immediately after HI, then 1 mg/kg maintenance
dose at 12 h intervals for 48h; topiramate group received
40 mg/kg loading dose then 10 mg/kg maintenance dose on
the same schedule as memantine; combination group
received both memantine and topiramate, the drug doses
and schedule were the same as above.
4.3. Gross brain damage grading
To quantify the severity of brain damage, rat pups were
decapitated at 22d after HI and their brains were rapidly
dissected and frozen (Uhm et al., 2003). Then brains were
scored normal, mild, moderate or severe by a blinded observer
according to the method of Palmer et al. (1990). The neurologic
damage scores were given according to the following criteria.
Normal (1) is no reduction in the size of the right hemisphere,
mild (2) is visible reduction in right hemisphere size, moderate
(3) is large reduction in hemisphere size from a visible infarct
in the right parietal area and severe (4) is near total destruction
of the hemisphere.
To measure the loss of hemispheric weight, the brain was
divided into two hemispheres and weighed after removing the
cerebellum and brainstem. Results are presented as the
percent loss of hemispheric weight of the right side relative
to the left [(left−right)/left×100]. The HI model used in this
study results in brain damage only on the ipsilateral side, thus
the loss of hemispheric weight can be used as a measure of
brain damage in this model (Rice et al., 1981). Because the
brain weighs approximately 1 g/ml, weight loss is equivalent
to volume loss. According to the method by McDonald et al.,
the loss of brain weight on the ipsilateral side relative to the
contralateral side is highly correlated with cellular damage
(McDonald et al., 1989b). For short, weighing can assess the
degree of brain damage.
4.4. Microscopic brain damage grading
Microscopic examination of the tissues was carried out to
verify that the gross changes were a reflection of the expected
histopathologic changes. The rat pups were anesthetized
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